Proteinuria is a cardinal sign of chronic kidney disease (CKD), which is a major health-care problem that affects millions of people worldwide. Recent advances in molecular genetics and cell biology have revealed the podocyte as the primary functional regulator of the tri-layered glomerular filter. Since podocyte foot processes (FP) and their interposed slit diaphragm (SD) form the final barrier to protein loss, podocyte injury leads to proteinuric kidney disease. The SD is a specialized dynamic cell-cell junction and contains several proteins that are also found in tight junctions (TJ) of classic epithelial cells Since structural features of the SD and TJ are different, the function of TJ proteins in podocytes is unclear, but most likely involves the regulation of SD function. The serum protein zonulin has been shown to induce TJ disassembly in epithelial cells of the small intestine thereby increasing paracellular flux of macromolecules across the epithelial barrier in a regulated and reversible manner. Zonulin activates protease-activated receptor (PAR) 2 and induces signaling cascades that regulate the junction protein zonula occludens (ZO)-1 and the actin cytoskeleton. Since podocytes express PAR2 and changes in ZO-1 phosphorylation and actin dynamics are established mechanisms underlying podocyte plasticity, we hypothesize that zonulin also functions as a reversible regulator of the podocyte SD and paracellular permeability in the glomerulus. Furthermore, we postulate that aberrant long-term elevation of serum zonulin leads to irreversible podocyte injury, proteinuria and eventually progressive kidney diseases. Our preliminary in vitro experiments show that zonulin treatment induces changes in the podocyte actin cytoskeleton in a PAR2-dependent manner, and also alters cell migration, ZO-1 phosphorylation and paracellular flux. Furthermore, zonulin increases albumin permeability of isolated rat glomeruli. Our in vivo experiments illustrate that mice on a high gluten diet, which increases serum zonulin levels, develop mild proteinuria. The goals for our proposed study are the following: First, we want to determine if zonulin-induced changes in the SD and paracellular permeability are PAR2-dependent. Therefore, we will replicate our in vitro studies in PAR2 knockdown podocytes, as well as in wild type podocytes in the presence of a PAR2-specific blocking peptide. Second, we will analyze if zonulin-mediated activation of PAR2 in podocytes leads to glomerular damage and albuminuria in vivo. We will analyze potential kidney phenotypes in an established transgenic mouse model with elevated serum zonulin. In parallel, we will inject a peptide mimicking zonulin's PAR2 binding site into wild type mice, and into a newly generated podocyte-specific PAR2 knockout mouse model. Since serum zonulin levels have been recently shown to be elevated in patients with CKD, and other circulating PAR2 activators like hemopexin and serine proteases have been linked to nephrotic syndrome, it is possible that PAR2 activation on podocytes is involved in a variety of glomerular diseases and that patients with nephrotic syndrome would benefit from pharmacological PAR2 inhibition.